Il-6 detection based early diagnosis and prediction of systematic inflammatory response syndrome and sepsis in asymptomatic patients

ABSTRACT

Methods, systems and kits for the early diagnosis or prediction of systemic inflammatory response syndrome (SIRS) including sepsis in asymptomatic patients, such as patients undergoing a surgical intervention, are provided. Some embodiments include a method and system for the detection or diagnosis of SIRS, or detection or diagnosis of a risk to suffer from or develop SIRS, in an asymptomatic patient comprising the steps of determining the level of IL-6 (or a variant thereof) in a sample from the patient; comparing the level of IL-6 (or a variant thereof) to a reference level; detecting or diagnosing SIRS or diagnosing a risk to suffer from or develop SIRS, wherein the sample is isolated at least 2 times at short intervals and the determining and comparing steps are both repeated for each sample. Also provided are methods, systems and kits for therapy monitoring and mortality prediction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2011/001006, filed Mar. 2, 2011, which claims the benefit ofEuropean Patent Application No. 10002190.6, filed Mar. 2, 2010, thedisclosures of which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE DISCLOSURE

Systemic inflammatory response (SIRS) and sepsis represent a common anddevastating syndrome that is increasing in frequency around the world.SIRS and sepsis are among the most frequent causes of death in intensivecare patients.

In 1991, the American College of Chest Physicians (ACCP) and the Societyof Critical Care Medicine (SCCM) provided a conceptual and practicalframework to define the systemic inflammatory response to infection(American College of Chest Physicians/Society of Critical Care MedicineConsensus Conference: definitions for sepsis and organ failure andguidelines for the use of innovative therapies in sepsis. Crit Care Med.1992 June;20(6):864-74). Uniform definitions for the various stages ofsepsis which could be universally and uniformly applied to patientssuffering from these disorders were also provided by the joint ACCP andSCCM Conference. Prior to this time, various terminologies were usedinterchangeably, leading to much confusion.

At the time point when the clinical signs of SIRS and sepsis appear, thedevelopment and aggravation of the SIRS has already begun. However, nocurrent methods exist for diagnosing SIRS and sepsis, or for predictingthe risk to suffer from or to develop SIRS and sepsis, prior to theonset of such generally recognized clinical signs and symptoms of SIRSand sepsis (for example, in an asymptomatic patient).

BRIEF SUMMARY OF THE DISCLOSURE

The present disclosure provides early diagnosis and prediction ofsystemic inflammatory response syndrome (SIRS) or sepsis in asymptomaticpatients, such as in patients undergoing a surgical intervention, usingII-6 as a diagnostic and predictive biomarker. Corresponding therapymonitoring and mortality prediction methods, kit of parts are alsoprovided. The diagnostic methods and treatment monitoring methodsprovided herein allow for a sensitive and early detection of SIRS orsepsis and for predicting the risk to suffer from or develop SIRS andsepsis, before the onset of clinical signs and symptoms of SIRS.

The present disclosure also provides methods and treatments formonitoring method, both of which enable early detection of the risk tosuffer from or develop SIRS and sepsis before the onset of clinicalsymptoms. The instant disclosure also provides a kit and a computerprogram adapted for carrying out these methods.

According to a first embodiment, it is provided a method for detectionor diagnosis of a systemic inflammatory response syndrome (SIRS) orsepsis, or for detection or diagnosis of a risk to develop or sufferfrom SIRS or sepsis, in an asymptomatic patient, comprising the steps ofdetermining the level of IL-6 or a variant thereof in a sample from thepatient, comparing the level of IL-6 or a variant thereof determined instep a) to a reference level and detecting or diagnosing SIRS, ordetecting or diagnosing a risk to develop or suffer from SIRS or sepsis.In some such embodiments, the sample may be isolated at least 2 times atshort intervals and the steps of determining and comparing are repeatedfor each sample.

In some embodiments, it is provided a method for detecting the level ofIL-6 in an asymptomatic patient for detection or diagnosis of a risk todevelop or suffer from SIRS or sepsis, comprising the steps ofdetermining the level of IL-6 or a variant thereof in a sample from thepatient and comparing the level of IL-6 or a variant thereof determinedin the determining step to a reference level. According to variousembodiments, based on the comparison it is detected or diagnosed if thepatient is at risk to develop or suffer from SIRS or sepsis. In someembodiments, the sample is isolated at least 2 times at short intervalsand the determining and comparing steps are repeated for each sample.

Yet in further embodiments of the instant disclosure, it is alsoprovided a method for detection or diagnosis of a risk to develop orsuffer from SIRS or sepsis, in an asymptomatic patient selected from atrauma patient, a patient with burns, a patient undergoing an invasivetreatment, a patient undergoing a surgery, comprising the steps ofdetermining the level of IL-6 or a variant thereof in a sample from thepatient and comparing the level of IL-6 or a variant thereof determinedin the step of determining to a reference level. The method alsoincludes detecting or diagnosing SIRS, or detecting or diagnosing a riskto develop or suffer from SIRS. The sample is isolated at least 2 timesat short intervals and the determining and comparing steps are repeatedfor each sample. In some embodiments, at least one sample is isolatedupon admission of the patient and at least one sample is isolated aftera treatment has been initiated or terminated.

In some embodiments of the above described embodiments, if theasymptomatic patient will be subjected to a invasive treatment at leastone sample is isolated before the surgical intervention or to obtain abaseline IL-6 level, then at least one further sample is isolated andanalyzed for IL-6 levels at short intervals after the completion of theinvasive treatment.

Additionally, embodiments of the instant disclosure include IL-6 or ameans for detecting IL-6 used to detect or diagnose the risk to developor suffer from SIRS or sepsis, in an asymptomatic patient, wherein thelevel of IL-6 is determined at least 2 times at short intervals.

BRIEF DESCRIPTION OF THE FIGURES

The features of this disclosure, and the manner of attaining them, willbecome more apparent and the disclosure itself will be better understoodby reference to the following description of embodiments of thedisclosure taken in conjunction with the accompanying drawing.

FIGS. 1 a-1 f If are a graph presenting IL-6 (pg/ml) kinetics inasymptomatic patients pre- and post-surgery showing a comparison betweenpooled patients who developed SIRS/sepsis and those who did not (havingIL-6 levels plotted for baseline 1 (pre-surgery), baseline 2 (duringsurgery), and post-surgery (day 1, 2, 3 and 4 in which samples weretaken at 6 hour intervals) and having Median and the percentilesindicated).

FIG. 2 a is a graph presenting the concentration of IL-6 (pg/ml) plottedover time for SIRS patient No. 1 (with the darkened triangle indicatingthe time point clinical SIRS signs were diagnosed).

FIG. 2 b is a graph presenting the concentration of IL-6 (pg/ml) plottedover time for SIRS patient No. 2 (with the darkened triangle indicatingthe time point clinical SIRS signs were diagnosed).

FIG. 2 c is a graph presenting the concentration of IL-6 (pg/ml) plottedover time for SIRS patient No. 3.

FIG. 2 d is a graph presenting the concentration of IL-6 (pg/ml) plottedover time for SIRS patient No. 4 (with the darkened triangle indicatingthe time point clinical SIRS signs were diagnosed).

FIG. 2 e is a graph presenting the concentration of IL-6 (pg/ml) plottedover time for SIRS patient No. 5 (with the darkened triangle indicatingthe time point clinical SIRS signs were diagnosed).

FIG. 2 f is a graph presenting the concentration of IL-6 (pg/ml) plottedover time for SIRS patient No. 6 (with the darkened triangle indicatingthe time point clinical SIRS signs were diagnosed).

FIG. 2 g is a graph presenting the concentration of IL-6 (pg/ml) plottedover time for SIRS patient No. 7 (with the darkened triangle indicatingthe time point clinical SIRS signs were diagnosed).

FIG. 3 is a graph presenting the median concentration of IL-6 (pg/ml)plotted over time for non-SIRS patients.

FIGS. 4 a-4 d are a graph presenting CRP (mg/L) kinetics in asymptomaticpatients pre- and post-surgery showing a comparison between pooledpatients who developed SIRS/sepsis and those who did not (having CRPlevels plotted for baseline 1 (pre-surgery), baseline 2 (duringsurgery), and post-surgery (day 1, 2, 3 and 4 in which samples weretaken at 6 hour intervals) and having Median and the percentilesindicated).

FIG. 5 a is a graph presenting a comparison of the CRP kinetics (mg/L)between pooled patients who developed SIRS/sepsis and those who did not(based on asymptomatic patients pre- and post-surgery).

FIG. 5 b is a graph presenting a comparison of the IL-6 kinetics (pg/ml)between pooled patients who developed SIRS/sepsis and those who did not(based on asymptomatic patients pre- and post-surgery).

Corresponding reference characters indicate corresponding partsthroughout the several views. Although the drawings representembodiments of the present disclosure, the drawings are not necessarilyto scale and certain features may be exaggerated in order to betterillustrate and explain the present disclosure. The exemplifications setout herein illustrate an exemplary embodiment of the disclosure, in oneform, and such exemplifications are not to be construed as limiting thescope of the disclosure in any manner.

DETAILED DESCRIPTION OF THE DISCLOSURE

The embodiments disclosed herein are not intended to be exhaustive orlimit the disclosure to the precise form disclosed in the followingdetailed description. Rather, the embodiments are chosen and describedso that others skilled in the art may utilize their teachings.

A number of conditions associated with a risk of developing sepsis andsevere sepsis have been identified through epidemiological evaluation.These factors include: gender (men); race (black); ethnicity (Hispanic);advanced age; and co-morbidities Diabetes mellitus, malignancy,alcoholism, HIV infection, and treatment with immunosuppressive agents(Hodgin K E, Moss M., The epidemiology of Sepsis. Current Pharm Design,2008;14:1833-1839, the disclosure of which is hereby incorporated byreference in its entirety). In addition, an event such as a majorsurgery, trauma, or burn which results in large area wounds alsopresents an additional risk. SIRS without infection may also occur inevents such as pancreatitis, shock, ischemia, and polytrauma.

IL-6 belongs to a family of gp130 cytokines. All family members share afour-helical protein structure and exert a signal via a receptor complexcontaining at least one subunit of the signal transducing receptorglycoprotein gp130. IL-6 binds to the IL-6 receptor (IL-6R) first andthis IL-6/IL-6R complex binds to gp130 leading to a homodimerization andsubsequent activation of the Jak/Stat- and Ras/Map/Akt-signaltransduction pathway. There are two different signaling pathways, one inwhich IL-6 binds to the membrane bound IL-6 receptor which leads todimerization and activation of the signal transducing protein gp130.This pathway is restricted to cells which express the IL-6 receptor ontheir surface, which is only the case in some cell populations. Howeveran alternative pathway exists in which IL-6 binds to a naturallyoccurring soluble IL-6R (sIL-6R) and this IL-6/sIL-6R complex activatesgp130. This way, cells lacking the membrane bound IL-6-receptor canrespond to IL-6. This second so called trans-signaling pathway alsoaffects cells which express membrane-bound IL-6 receptors (for example,hepatocytes). In this setting an activation of IL-6 trans-signaling canenhance stimulatory effects of IL-6.

IL-6 has shown activity in both B-cells, T-cells, hematopoietic stemcells, hepatocytes and brain cells. Body fluids of patients with localacute infection and serum of patients with gram-negative andgram-positive bacteraemia have been shown to contain elevated levels ofbiologically active IL-6. Also, IL-6 presence serum during acuteinfection has been shown (suggesting this cytokine may participate inboth local and systemic events for limiting tissue damage).

However, diagnosing SIRS and sepsis, predicting the risk to suffer fromor to develop SIRS and sepsis, before the onset of generally recognizedclinical signs and symptoms of SIRS and sepsis (for example, in anasymptomatic patient) has not been demonstrated or suggested. Thepresent disclosure provides a surprising and unexpected method andsystem for a diagnostic method and a treatment monitoring methodallowing for a sensitive and early detection of SIRS or sepsis and forpredicting the risk to suffer from or develop SIRS and sepsis, beforethe onset of clinical signs and symptoms of SIRS.

The present disclosure also provides means and methods which solve atleast some of the shortcomings of the hitherto known approaches todiagnosing SIRS and sepsis. Moreover, the methods and treatmentsprovided include monitoring methods which allow for an early detectionof the risk to suffer from or develop SIRS and sepsis before the onsetof clinical symptoms. Further, the present disclosure provides kits andcomputer programs adapted for carrying out these methods.

The instant disclosure sets forth a method and system which provides thesurprising and unexpected result of allowing for a clear identificationof asymptomatic patients at risk of suffering from or developing SIRSincluding sepsis well before the onset of clinical signs or pathologicalchanges of laboratory parameters conventionally used to diagnoseSIRS/sepsis (see e.g. Examples). According to the instant disclosure,close-meshed consecutive measurements of IL-6, for example beginningbefore a treatment (such as a therapy associated with a significant riskof resulting in SIRS or sepsis) such as a major surgery, allows for aclear identification of asymptomatic patients at risk of suffering fromor developing SIRS including sepsis well before the onset of clinicalsigns or pathological changes of laboratory parameters conventionallyused to diagnose SIRS/sepsis. The instant disclosure sets forth, for thefirst time, IL-6 kinetics in asymptomatic patients which can be usefulin identifying such patients as developing (or at risk for developing)SIRS. As disclosed herein, a large rise in IL-6 concentration whencompared to a baseline level indicates a high risk to develop or sufferfrom SIRS or sepsis, well ahead of the display of clinical symptoms orsigns supporting the diagnosis of SIRS and sepsis. Thus, contrary towhat was known before, rather than analyzing the absolute level of IL-6in the sample, the diagnosis of the risk to suffer from or develop SIRSor sepsis is essentially based on determining the increase of the IL-6level over time. Furthermore, the early response properties of themethod of the present disclosure enables, prior to the appearance ofclinical signs and symptoms of SIRS and sepsis, the ability to initiateappropriate treatments at an earlier point in time, when compared tohitherto known method of diagnosing SIRS and sepsis. The instantdisclosure further provides methods and systems which enhance thelikelihood of a successful therapy to SIRS, or the prevention of SIRS orsepsis, and therefore provides for the improvement of treatments ofpatients against SIRS or sepsis.

As used herein, the phrase “systemic inflammatory response syndrome(SIRS)” is generally known to the skilled worker. According to theinstant disclosure, this phrase encompasses SIRS as defined on theACCP/SCCM Consensus Conference Definitions (1992/2003) (see e.g.American College of Chest Physicians/Society of Critical Care MedicineConsensus Conference: definitions for sepsis and organ failure andguidelines for the use of innovative therapies in sepsis. Crit Care Med.1992 June;20(6):864-74)). A patient is generally considered to sufferfrom SIRS, if the patient displays at least 2 symptoms of the following:

-   -   a) white blood cell count (WBC)>about 12,000/μ/L or <about        4000/μ/L;    -   b) body temperature >about 38° C. or <about 36° C.;    -   c) heart rate >about 90 beats/minute;    -   d) a respiratory rate >about 20 breaths/minute or a partial        pressure of CO₂ of less than about 32 mm Hg; and    -   e) more than 10% immature white blood cells among the counted        white blood cells.        At the time point when the clinical signs appear, the        development and aggravation of the SIRS has already been        started.

White blood cell counts may generally be determined by automatedcounting devices, for example, as known in the art.

With regard to children, the consensus criteria for diagnosing SIRS in achild are disclosed in Goldstein et al. (Pediatr. Crit Care Med 2005,6(1), 2-8, see in particular Tables 2 and 3, the disclosure of which ishereby incorporated by reference in its entirety). An asymptomatic childpatient in the sense of the present disclosure is a patient displayingless than 2, and in some cases less than 1 symptom of the thosedescribed in Goldstein et al.

As disclosed and described herein, the risk of suffering from ordeveloping SIRS may be diagnosed or detected on the basis of an IL-6level detected in a sample of a patient which is above the referencelevel. According to embodiments of the instant disclosure, the detectionand diagnosis set forth herein do not require the patient to display atleast two of the above SIRS symptoms (a) to (e). As disclosed herein,the risk to suffer from or develop SIRS or sepsis may be diagnosed ordetected solely on the basis of a determined IL-6 level which is abovethe reference level. In some embodiments, the reference value isdetermined by multiplying an earlier determined IL-6 level (e.g. abaseline IL-6 level) in a given patient by a factor defined elsewhereherein (e.g. a factor of at least about 50, at least about 100, at leastabout 500, and in some embodiments at least about 1000). According toembodiments of the instant disclosure, IL-6 levels determined in one ormore samples isolated from the patient after the earlier sample(s)(which provide the baseline level) was collected, and which compriseIL-6 levels above the reference level are indicative of the patient tobe at a high risk to develop or suffer from SIRS. The present disclosureprovides that, based on the IL-6 determinations disclosed and taughtherein, early detection and diagnosis of the risk to suffer from ordevelop SIRS is possible, even before the patient displays two or moreof the above SIRS symptoms (a) to (e).

As used herein, for “sepsis” the diagnostic criteria mentioned for“SIRS” above applies mutatis mutandis, however, in sepsis, a diagnosedinfection is a additional diagnostic parameter generally required.Methods for the detection or diagnosis of an infection are generallyknown in the field. The present disclosure provides that the risk tosuffer from or develop sepsis may now be detected or diagnosed on thebasis of only two parameters, i.e. a level of IL-6 above the referencelevel and a diagnosed infection.

An “infection” in the sense of the present disclosure may be viral,fungal or bacterial infection. According to some embodiments, theinfection may be a bacterial infection associated with bacteria selectedfrom E coli, staphylococcus aureus, Klebsiella pneumoniae, Streptococcior Pseudomonas aeroginosa. Also, by way of example, the infection mayalso be an infection by a fungus selected from Candida albicans, Candidatropicalis or Aspergillus fumigatus, for example.

An infection is diagnosed on the basis of assays and criteria generallyknown to the physician. For example, the infection may be diagnosed onthe basis of a bacterial culture assay, e.g. a culture medium inoculatedwith a sample from the patient, or based on molecular diagnosticmethods. A fungal infection may, for example, be determined based on thegenerally known test assays such as Septifast.

The term “patient” as used herein relates to animals, including mammalssuch as but not limited to humans, dogs, cats, horses, and cattle.Humans include male or female, and also includes prenatal, perinatal,postnatal or neonatal children.

As used herein, the expression “asymptomatic patient” encompasses apatient not displaying clinical signs and symptoms generally consideredto establish diagnosis of SIRS. For example, the asymptomatic patient ingeneral will display less than 2 symptoms and in some cases, less than 1symptom of the following:

-   -   a) white blood cell count (WBC) >about 12,000/μ/L or <about        4000/μ/L;    -   b) body temperature >about 38° C. or <about 36° C.;    -   c) heart rate >about 90 beats/minute;    -   d) a respiratory rate >about 20 breaths/minute or a partial        pressure of CO₂ of less than about 32 mm Hg; and    -   e) more than 10% immature white blood cells among the counted        white blood cells.

The instant disclosure is also applicable with a asymptomatic patientswhich include trauma patients, patients with burns, patients undergoingan invasive treatment such as a surgical intervention (including andendoscopic intervention), and patients selected from findings on aCT-scan or PET-CT-scan. The instant disclosure is further applicablewith asymptomatic patients having a risk of developing SIRS or sepsissuch as a patient meeting at least one of the following criteria:

-   -   genetic disposition for sepsis;    -   premature (perinatal, neonatal) or advanced age;    -   sex is male;    -   race is African American;    -   medical co-morbidities including a chronic illness (like        diabetes, congestive heart failure), pre-existing organ        dysfunction (like cirrhosis or renal failure), physical or        mental impairment;    -   previous clinical interventions (like major surgery,        endotracheal intubation, antibiotics); and    -   social, religious or cultural factors.

The aforementioned criteria are described in further detail in MarshallJ C (“Predisposition to Sepsis”, Anaesthesia, Pain, Intensive Care andEmergency A.P.I.C.E., Springer Verlag, ISBN 88-470-0772-0, thedisclosure of which is hereby incorporated by reference in itsentirety). These factors may be taken into account when practicing themethods and systems of the present disclosure.

According to some embodiments, asymptomatic patients may exclude apatient where the metabolization of IL-6 is impaired (for example,patients where IL-6 clearance via the splanchnic and kidney organ isimpaired as discussed in Garibotta et al., Cytokine, 2007, 37, 51-54,the disclosure of which is hereby incorporated by reference in itsentirety).

According to the present disclosure, “interleukin-6 (IL-6)” encompassesIL-6, as it is generally known in the art. For example, IL-6 encompassesinterferon-Î²2, plasmacytoma growth factor, hepatocyte stimulatingfactor and human B-cell-stimulating factor 2 (BSF2). IL-6, as usedherein, may also comprise a protein produced from a single gene encodinga product of 212 amino acids, or ins some cases a product of 184 aminoacids of the IL-6 peptide which is cleaved at the N-terminus of the 212amino acid peptide (see Song M, Kellum J A. Interleukin-6. Crit Care Med2005; 33 (Suppl12): 463-465 and NCBI sequence for the 212 amino acidlong IL-6 precursor, accession number NP_(—)000591, the disclosure ofwhich is hereby incorporated by reference in its entirety). IL-6 alsoencompasses free IL-6 which is not bonded to its receptor IL-6R.Moreover, IL-6 may also encompass IL-6 in the state of the IL-6/IL-6Rcomplex (see Taga T, Kishimoto T. Gp130 and the Interleukin-6 Family ofCytokines. Annu. Rev. Immunol. 1997; 15: 797-819; Drucker C, Gewiese J,Malchow S, Scheller J, Rose-John S. Impact of Interleukin-6 Classic- andTrans-signaling on Liver Damage and Regeneration. J Autimm 2009 inpress). IL-6 may also comprise the IL-6 protein which can be bound orwhich is bound by the monoclonal anti-IL6 antibody M-BE8 (as defined inEP0430193, i.e. an antibody produced by the cell line BE-8, or in KLEIN,B., et al. 1991, Murine anti-interleukin 6 monoclonal antibody therapyfor a patient with plasma cell leukemia, Blood 78, 1198-1204) or M-23C7.Also, IL-6 may comprise the IL-6 which can be bound or which is bound bythe antibody of Roche's IL-6 assay for use on Elecsys and cobasimmunoassay systems (Roche). As used herein, IL-6 also encompasses avariant of the aforementioned IL-6, such as human IL-6. The variantencompasses a protein or peptide substantially similar to the specificreference IL-6 molecule, such as the human IL-6. As used herein, theterm substantially similar is well understood by the person skilled inthe art.

Some embodiments of the instant disclosure may comprise an IL-6 variant.According to the present disclosure and IL-6 variant may be an isoformor allele which shows at least one amino acid exchange includingsubstitutions, additions and deletions (for example, in some cases up toabout 3, up to about 5, up to about 10, up to about 15, and even up toabout 25 amino acid exchanges) compared to the amino acid sequence ofthe specific reference IL-6 molecule. In some embodiments, an IL-6variant has a sequence identity to the specific reference IL-6 moleculeof at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 98%, with respect to human IL-6. Suchidentity, according to some embodiments, is over the entire length ofthe human IL-6. The degree of identity between two amino acid sequencescan be determined by algorithms well known in the art, for example. Ingeneral, the degree of identity is determined by comparing two optimallyaligned sequences over a comparison window, where the fragment of aminoacid sequence in the comparison window may comprise additions ordeletions (e.g., gaps or overhangs) as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment. The percentage can be calculated, for example, by determiningthe number of positions at which the identical amino acid residue occursin both sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Examples of algorithm for optimalalignment of sequences for comparison may be conducted by the localhomology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981),by the homology alignment algorithm of Needleman and Wunsch J. Mol.Biol. 48:443 (1970), by the search for similarity method of Pearson andLipman Proc. Natl. Acad Sci. (USA) 85: 2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG), 575 Science Dr., Madison, Wis.). Alignment of sequences ofcomparison may also be accomplished by visual inspection. Given that twosequences have been identified for comparison, GAP and BESTFIT may beemployed to determine their optimal alignment and, thus, the degree ofidentity. In such embodiments, default values of 5.00 for gap weight and0.30 for gap weight length may be used.

Variants as referred to above (and herein) may be allelic variants orany other species specific homologs, paralogs, or orthologs. Theexpression variant also encompasses degradation products, e.g.proteolytic degradation products, which are still recognized by thediagnostic means or by ligands directed against the respectivefull-length protein or peptide. The term “variants” is also meant tocover splice variants and also relates to a post-translationallymodified peptide such as glycosylated peptide. A “variant” is also apeptide which has been modified after collection of the sample, forexample by covalent or non-covalent attachment of a label, such as aradioactive or fluorescent label, to the peptide. In some embodiments,the IL-6 variant possesses essentially the same immunological and/orbiological properties of the specific reference peptide, which in somecases is human IL-6. Also, according to various embodiments, the IL-6variant may display at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, or at least about 98% of the human IL-6activity. The IL-6 activity according to some embodiments is defined asthe IL-6 receptor binding activity (see Taga T, Kishimoto T. Gp130 andthe Interleukin-6 Family of Cytokines. Annu. Rev. Immunol. 1997; 15:797-819; Drucker C, Gewiese J, Malchow S, Scheller J, Rose-John S.Impact of Interleukin-6 Classic- and Trans-signaling on Liver Damage andRegeneration. J Autimm 2009 in press.). The IL-6 variant, according tosome embodiments, displays a human IL-6 receptor binding activity of atleast about 80%, at least about 90%, or at least about 95% of humanIL-6.

According to a some embodiments of the present disclosure at least oneadditional marker or parameter indicative of SIRS or sepsis isdetermined including at least one parameter selected from aninflammation marker like CRP, another interleukin like IL-1 and/or IL-8,procalcitonin, white blood cell count, body temperature, heart rate,respiratory rate, and/or diagnosis of an infection, preferably abacterial and/or fungus infection.

Moreover, the method of the present disclosure may comprise steps inaddition to those explicitly mentioned above. For example, further stepsmay relate to sample collection, sample pre-treatments or evaluation ofthe results obtained by the method. The method of the present disclosuremay be also used for monitoring, confirmation, sub-classification andrisk assessment of developing or suffering from SIRS and for therapeuticmonitoring the diseases of the present disclosure. It is also envisionedthat in some embodiments at least one additional marker or parameter maybe determined apart from the two mentioned in step a), such as for thepurpose of obtaining additional diagnostic information beyond thedetection or diagnosis of SIRS or sepsis. Such additional parameter mayfor example be the estimated glomerular filtration rate, the level ofNGAL or creatinine which allows to additionally diagnose if the patientsuffers from an impaired renal clearance.

The method may be carried out manually and/or assisted by automation.For example, steps (a), (b), and/or (c) may in total or in part beassisted by automation, e.g., by a suitable robotic and sensoryequipment for the determination in step (a) or a computer-implementedcomparison in step (b) and/or (c).

The term “sample” as used herein refers to a sample of a body fluid, toa sample of separated cells, or to a sample from a tissue or an organ.Samples of body fluids can be obtained by well-known techniques andinclude, but are not limited to, samples of blood, plasma, serum, liquoror urine. In some cases, body fluid samples are obtained byvenopuncture, arterial puncture or ventricular puncture. Tissue or organsamples may be obtained from any tissue or organ by, for example,biopsy. Separated cells may be obtained from the body fluids or thetissues or organs by separating techniques such as centrifugation orcell sorting. In some embodiments, cell-, tissue- or organ samples areobtained from those cells, tissues or organs which express or producethe peptides referred to herein.

Further, the methods of the present disclosure may encompass a step ofcollecting a sample, which optionally may be an invasive step. However,the sample may be collected by way of a minimal invasive step, such asby venopuncture, as well. The minimal invasive collection alsoencompasses the case where the sample is collected by use of a needle(lancette) which when applied to the skin (for example the skin of afinger) elicits outflow of a small volume of blood which may then becollected for determining the amount of the markers in the sample.

The present disclosure, according to some embodiments, consists of an exvivo or in vitro method. According to such embodiments, the samples maybe isolated at short intervals from a patient. As used herein, “shortintervals” encompasses an interval ranging from about 15 minutes toabout 12 hours. The samples, according to some embodiments are isolatedfrom the patient for a period of up to about 10 days (although invarious embodiments the period may be up to only about 2 or about 3days). Depending on the condition and development of the condition ofthe patient the isolation of the samples and IL-6 detection may befurther prolonged, for example, if the patient develops SIRS or sepsisthe sampling may be extended until the SIRS or sepsis therapy has beensuccessfully completed or even a few days beyond that point in time.According to the instant disclosure the samples are taken at least once,and in some cases at least about twice before a treatment is carriedout, and then at least once or at least about twice after the treatment.For example, prior to the treatment being carried out, one or two IL-6levels may be taken. Following the treatment a number of samples may betaken at regular intervals such as 3-6 hours over a period of about 3 to10 days, where IL-6 (and in some embodiments also the clinical status)of the patient is determined.

The phrase “detecting and diagnosing SIRS” as used herein meansassessing, identifying, evaluating, or classifying if an asymptomaticpatient suffers from SIRS, preferably from sepsis.

The phrase “detecting and diagnosing a risk to develop or suffer fromSIRS” is meant to encompass the prediction of the risk in anasymptomatic patient to develop or to suffer from SIRS, includingsepsis, within a defined time window (predictive window) in the future.The predictive window is an interval in which the subject will develop aSIRS or optionally will die according to the predicted probability. Insome cases, the predictive window is an interval of up to about 20 days,but may be up to about 10 days or less such as about 7 days, about 5days, about 4 days, about 3 days, or about 2 days, (i) after the methodof the present disclosure has been carried out or (ii) after the firstsample was obtained in which the detected IL-6 level was above thereference level or (iii) after admittance of the patient or (iv) afterthe first baseline level for IL-6 has been obtained/isolated or (v)after the treatment (e.g., the invasive treatment) has been initiated orterminated, or (vi) after the first post-treatment sample has beenisolated for determination of the IL-6 level.

Patients identified as being at risk of developing or suffering fromSIRS, according to the instant disclosure, may possess a highprobability of developing or suffering from SIRS. In general, the highprobability will be at least about 30% or more (for example, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%). Optionally,a high probability of developing or suffering from SIRS or sepsis,encompasses a probability of 100%, i.e. the patient will develop or doessuffer from SIRS or sepsis.

Alternatively, the patient may have a low probability of suffering fromor developing SIRS or sepsis. For example, the low probability may be upto about or less than about 30%, or up to about 20%, up to about 10%, orup to about 5%. Optionally, a low probability of developing or sufferingfrom SIRS or sepsis, encompasses a probability of 0%, i.e. the patientwill not develop or does not suffer from SIRS or sepsis.

Methods for the determination of the % risk of suffering from ordeveloping SIRS or sepsis are generally known. By way of example, the %risk prediction may be carried out within the predictive windowmentioned above.

According to the instant disclosure, the extent of the risk to developor suffer from SIRS or sepsis may correlate with the extent the detectedIL-6 level is above the reference value. Likewise, the risk ofdeveloping or suffering from SIRS or sepsis may be low in cases wherethe detected IL-6 level is lower than the reference level. Further, atleast according to some embodiments, the risk of developing or sufferingfrom SIRS or sepsis is high in cases where the detected IL-6 level isequal to or above the reference value.

As will be understood by those skilled in the art, such an assessment isusually not intended to be correct for 100% of the subjects to beanalyzed. The term, however, requires that the assessment will be validfor a statistically significant portion of the subjects to be analyzed.Whether a portion is statistically significant can be determined by theperson skilled in the art using any of various well known statisticevaluation tools, including by not limited to, a determination ofconfidence intervals, p-value determination, Student's t-test,Mann-Whitney test, etc. Details of some tools may be found in Dowdy andWearden, Statistics for Research, John Wiley & Sons, New York 1983. Insome instances, confidence intervals are at least 90%, at least 95%, atleast 97%, at least 98% or at least 99%. The p-values are, in someembodiments, are 0.1, 0.05, 0.01, 0.005, or 0.0001. Also, theprobability encompassed by the present disclosure allows that theprediction will be correct for at least 60%, at least 70%, at least 80%,or at least 90% of the subjects of a given cohort.

Determining the level of IL-6 or a variant thereof, or of any otherproteinaceous biomarker according to the disclosure, relates tomeasuring the amount or concentration, including semi-quantitatively orquantitatively. Measuring can be done directly or indirectly. Directmeasuring relates to measuring the amount or concentration of thepeptide or polypeptide based on a signal which is obtained from thepeptide or polypeptide itself and the intensity of which directlycorrelates with the number of molecules of the peptide present in thesample. Such a signal, sometimes referred to herein as intensity signal,may be obtained by measuring an intensity value of a specific physicalor chemical property of the peptide or polypeptide, for example.Indirect measuring includes measuring of a signal obtained from asecondary component (i.e. a component not being the peptide orpolypeptide itself) or a biological read out system such as measurablecellular responses, ligands, labels, or enzymatic reaction products.

In accordance with the present disclosure, determining the amount of anIL-6 peptide or polypeptide can be achieved by all known means fordetermining the amount of a peptide in a sample. Said means compriseimmunoassay devices and methods which may utilize labeled molecules invarious sandwich, competition, or other assay formats. Said assaysgenerally develop a signal which is indicative for the presence orabsence of the peptide or polypeptide. Moreover, in some embodiments,the signal strength may be correlated directly or indirectly (e.g.reverse-proportional) to the amount of polypeptide present in a sample.Further suitable methods comprise measuring a physical or chemicalproperty specific for the peptide or polypeptide such as its precisemolecular mass or NMR spectrum. Said methods comprise biosensors,optical devices coupled to immunoassays, biochips, analytical devicessuch as mass-spectrometers, NMR-analyzers, or chromatography devices.Further, methods include micro-plate ELISA-based methods,fully-automated or robotic immunoassays (available for example onRoche's Elecsys™ analyzers), CBA (an enzymatic Cobalt Binding Assay,available for example on Roche-Hitachi™ analyzers), and latexagglutination assays (available for example on Roche-Hitachi™analyzers), homogenous and heterogeneous immune assays, competitive andnon-competitive immune assays.

According to various embodiments, determining the amount of an IL-6peptide or polypeptide comprises the steps of (a) contacting a cellcapable of eliciting a cellular response, the intensity of which isindicative of the amount of the peptide or polypeptide with the saidpeptide or polypeptide, for an adequate period of time, and (b)measuring the cellular response. For measuring cellular responses, thesample or processed sample is, in some embodiments, added to a cellculture and an internal or external cellular response is measured. Thecellular response may include the measurable expression of a reportergene or the secretion of a substance such as a peptide, polypeptide, ora small molecule. The expression or substance may generate an intensitysignal which correlates to the amount of the peptide or polypeptide.

Also according to embodiments of the instant disclosure, determining theamount of a IL-6 peptide or polypeptide comprises the step of measuringa specific intensity signal obtainable from the peptide or polypeptidein the sample, for example in a sample selected from blood, serum,plasma or liquor. As described above, such a signal may be the signalintensity observed at an m/z variable specific for the peptide orpolypeptide observed in mass spectra or a NMR spectrum specific for thepeptide or polypeptide.

Determining the amount of a IL-6 peptide or polypeptide may alsocomprise the steps of (a) contacting the peptide with a specific ligand,(b) (optionally) removing non-bound ligand, (c) measuring the amount ofbound ligand. The bound ligand will generate an intensity signal.Binding according to the present disclosure includes both covalent andnon-covalent binding.

A ligand according to the present disclosure can be any compound, e.g.,a peptide, polypeptide, nucleic acid, or small molecule, binding to thepeptide or polypeptide described herein. Exemplary ligands includeantibodies, nucleic acids, peptides or polypeptides such as receptors orbinding partners for the peptide or polypeptide and fragments thereofcomprising the binding domains for the IL-6 peptides, and aptamers, e.g.nucleic acid or peptide aptamers. Methods of preparing such ligands arewell-known in the art. For example, identification and production ofsuitable antibodies or aptamers is also offered by commercial suppliers.The person skilled in the art is familiar with methods to developderivatives of such ligands with higher affinity or specificity. Forexample, random mutations can be introduced into the nucleic acids,peptides or polypeptides. These derivatives can then be tested forbinding according to screening procedures known in the art, e.g. phagedisplay.

Means for the detection of IL-6 are generally known in the art andpreferably include anti-IL-6 antibodies, including polyclonal andmonoclonal antibodies, as well as fragments thereof, such as Fv, Fab andF(ab)2 fragments that are capable of binding IL-6 antigen or hapten. Themeans for the detection of IL-6 of the present disclosure also includesingle chain antibodies, chimeric, humanized hybrid antibodies whereinamino acid sequences of a non-human donor antibody exhibiting a desiredantigen-specificity are combined with sequences of a human acceptorantibody. Also included is an anti-IL-6 antibody from a mammalianspecies, such as an antibody selected from human, rat, mouse, goat,sheep, cattle, and camel. In some embodiments the anti-IL-6 antibody isthe M-BE8 or M-23C7 anti-IL-6 antibody described above or an antibodybinding to the IL-6 epitope recognized by the M-BE8 and/or M-23C7antibody. The donor sequences will usually include at least theantigen-binding amino acid residues of the donor but may comprise otherstructurally and/or functionally relevant amino acid residues of thedonor antibody as well. Such hybrids can be prepared by several methodswell known in the art. According to embodiments of the instantdisclosure, the ligand or agent binds specifically to the IL-6 peptideor polypeptide. Specific binding according to the present disclosuremeans that the ligand or agent should not bind substantially to(“cross-react” with) another peptide, polypeptide or substance presentin the sample to be analyzed. For example, the specifically bound IL-6peptide or polypeptide should be bound with at least 3 times higheraffinity than any other relevant peptide or polypeptide. In someembodiments of the instant disclosure, the specifically bound IL-6peptide or polypeptide may bind with at least at least 10 times higheror even at least 50 times higher affinity than any other relevantpeptide or polypeptide. Non-specific binding may be tolerable, if it canstill be distinguished and measured unequivocally, e.g. according to itssize on a Western Blot, or by its relatively higher abundance in thesample. Binding of the ligand can be measured by any method known in theart. Embodiments of the instant disclosure include said method beingsemi-quantitative or quantitative. Suitable methods are described in thefollowing.

First, binding of a ligand may be measured directly, e.g. by NMR orsurface plasmon resonance. Second, if the ligand also serves as asubstrate of an enzymatic activity of the peptide or polypeptide ofinterest, an enzymatic reaction product may be measured (e.g. the amountof a protease can be measured by measuring the amount of cleavedsubstrate, e.g. on a Western Blot). Alternatively, the ligand mayexhibit enzymatic properties itself and the “ligand/peptide orpolypeptide” complex or the ligand which was bound by the peptide orpolypeptide, respectively, may be contacted with a suitable substrateallowing detection by the generation of an intensity signal. Formeasurement of enzymatic reaction products, in some embodiments theamount of substrate is saturating. The substrate may also be labeledwith a detectable label prior to the reaction. According to embodimentsof the instant disclosure, the sample is contacted with the substratefor an adequate period of time. An adequate period of time refers to thetime necessary for a detectable (and in some cases measurable) amount ofproduct to be produced. Instead of measuring the amount of product, thetime necessary for appearance of a given (e.g. detectable) amount ofproduct can be measured.

Third, the ligand may be coupled covalently or non-covalently to a labelallowing detection and measurement of the ligand. Labeling may be doneby direct or indirect methods. Direct labeling involves coupling of thelabel directly (covalently or non-covalently) to the ligand. Indirectlabeling involves binding (covalently or non-covalently) of a secondaryligand to the first ligand. The secondary ligand should, in general,specifically bind to the first ligand. Said secondary ligand may becoupled with a suitable label and/or be the target (receptor) oftertiary ligand binding to the secondary ligand. The use of secondary,tertiary or even higher order ligands is often used to increase thesignal. Suitable secondary and higher order ligands may includeantibodies, secondary antibodies, and/or a streptavidin-biotin system(Vector Laboratories, Inc.). The ligand or substrate may also be“tagged” with one or more tags as known in the art. Such tags may thenbe targets for higher order ligands. Suitable tags include biotin,digoxygenin, His-Tag, Glutathion-S-Transferase, FLAG, GFP, myc-tag,influenza A virus haemagglutinin (HA), maltose binding protein, and thelike. In the case of a peptide or polypeptide, the tag is preferably atthe N-terminus and/or C-terminus. Suitable labels are any labelsdetectable by an appropriate detection method. Typical labels includegold particles, latex beads, acridan ester, luminol, ruthenium,enzymatically active labels, radioactive labels, magnetic labels (“e.g.magnetic beads”, including paramagnetic and superparamagnetic labels),and fluorescent labels. Enzymatically active labels include e.g.horseradish peroxidase, alkaline phosphatase, beta-Galactosidase,Luciferase, and derivatives thereof. Suitable substrates for detectioninclude di-amino-benzidine (DAB), 3,3′-5,5′-tetramethylbenzidine,NBT-BCIP (4-nitro blue tetrazolium chloride and5-bromo-4-chloro-3-indolyl-phosphate, available as ready-made stocksolution from Roche Diagnostics), CDP-Star™ (Amersham Biosciences), ECF™(Amersham Biosciences). A suitable enzyme-substrate combination mayresult in a colored reaction product, fluorescence or chemoluminescence,which can be measured according to methods known in the art (e.g. usinga light-sensitive film or a suitable camera system). As for measuringthe enyzmatic reaction, the criteria given above apply analogously.Typical fluorescent labels include fluorescent proteins (such as GFP andits derivatives), Cy3, Cy5, Texas Red, Fluorescein, and the Alexa dyes(e.g. Alexa 568). Further fluorescent labels are available e.g. fromMolecular Probes (Oregon). Also the use of quantum dots as fluorescentlabels is contemplated. Typical radioactive labels include 35S, 125I,32P, 33P and the like. A radioactive label can be detected by any methodknown and appropriate, e.g. a light-sensitive film or a phosphor imager.Suitable measurement methods according the present disclosure alsoinclude, for example, precipitation (particularly immunoprecipitation),electrochemiluminescence (electro-generated chemiluminescence), RIA(radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwichenzyme immune tests, electrochemiluminescence sandwich immunoassays(ECLIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA),scintillation proximity assay (SPA), turbidimetry, nephelometry,latex-enhanced turbidimetry or nephelometry, or solid phase immunetests. Further methods known in the art (such as gel electrophoresis, 2Dgel electrophoresis, SDS polyacrylamid gel electrophoresis (SDS-PAGE),Western Blotting, and mass spectrometry), can be used alone or incombination with labeling or other detection methods as described above.

According to embodiments of the present disclosure, the amount of IL-6is determined by mass spectrometry method, for example byisotope-dilution micro-HPLC-tandem mass spectrometry method, for exampleby a method as described in the Examples and in Kobold U et al. (ClinChem 2008; 54: 1584-6), hereby incorporated by reference in itsentirety.

The amount of an IL-6 peptide or polypeptide may also be determined asfollows: (a) contacting a solid support comprising a ligand for thepeptide or polypeptide as specified above with a sample comprising thepeptide or polypeptide and (b) measuring the amount peptide orpolypeptide which is bound to the support. The ligand, may be chosenfrom the group consisting of nucleic acids, peptides, polypeptides,antibodies and aptamers, and may be present on a solid support inimmobilized form. Materials for manufacturing solid supports are wellknown in the art and include, inter alia, commercially available columnmaterials, polystyrene beads, latex beads, magnetic beads, colloid metalparticles, glass and/or silicon chips and surfaces, nitrocellulosestrips, membranes, sheets, duracytes, wells and walls of reaction trays,plastic tubes etc. The ligand or agent may be bound to many differentcarriers. Examples of well-known carriers include glass, polystyrene,polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran,nylon, amyloses, natural and modified celluloses, polyacrylamides,agaroses, and magnetite. The nature of the carrier can be either solubleor insoluble for the purposes of the disclosure. Suitable methods forfixing/immobilizing said ligand are well known and include, but are notlimited to ionic, hydrophobic, covalent interactions and the like. It isalso contemplated to use “suspension arrays” as arrays according to thepresent disclosure (Nolan 2002, Trends Biotechnol. 20(1):9-12). In suchsuspension arrays, the carrier, e.g. a microbead or microsphere, ispresent in suspension. The array consists of different microbeads ormicrospheres, possibly labeled, carrying different ligands. Methods ofproducing such arrays, for example based on solid-phase chemistry andphoto-labile protective groups, are generally known (U.S. Pat. No.5,744,305).

The term “about” as used herein encompasses a range of + and −20%relative to the specific value, amount, concentration, level, etc, e.g.indication of a value of “about 100” is meant to encompass a value of anumerical range of 100+/−20%, i.e. a value range from 80 to 120. Forexample, the term “about” encompasses a range of + and −10% relative tothe specific value, amount, concentration, level, etc, and in someembodiments will encompass a range of + and −5% relative to the specificvalue, amount, concentration, level, etc.

The term “comparing” as used herein encompasses comparing the level ofthe IL-6 peptide or polypeptide comprised by the sample to be analyzedwith a level of a suitable reference source specified elsewhere in thisdescription. It is to be understood that comparing as used herein refersto a comparison of corresponding parameters or values, e.g., an absoluteamount is compared to an absolute reference level while a concentrationis compared to a reference concentration or an intensity signal obtainedfrom a test sample is compared to the same type of intensity signal of areference sample. The comparison referred to in step (b) of the methodof the present disclosure may be carried out manually or computerassisted. For a computer assisted comparison, the value of thedetermined level may be compared to values corresponding to suitablereferences which are stored in a database by a computer program. Thecomputer program may further evaluate the result of the comparison, i.e.automatically provide the desired assessment in a suitable outputformat. Based on the comparison of the level determined in step a) andthe reference level, the diagnosis of SIRS in a patient is determined.Therefore, the reference level is to be chosen so that either adifference or a similarity in the compared levels allows allocation ofsubjects in to SIRS or not suffering from SIRS.

Accordingly, the term “reference level”, refers to an amount,concentration or value which defines a cut-off. An amount, concentrationor value of the parameter above the cut-off results in a differentdiagnosis when compared to patients displaying a determined amount,level or value of the parameter below the cut-off. Thus, by comparingthe actually determined amount, concentration or value of the parametersIL-6, to the respective cut-offs it is possible to diagnose or detectthe risk in patients to develop or suffer from SIRS or sepsis.

Of course, the reference level applicable for an individual subject mayvary depending on various physiological parameters such as age, gender,subpopulation, alcohol intake, recent infections as well as on the meansused for the determination of the polypeptide or peptide referred toherein. A suitable reference level may be determined by the method ofthe present disclosure from a reference sample to be analyzed together,i.e. simultaneously or subsequently, with the test sample. The referencelevels of the present disclosure were confirmed in the Examples.

As will be understood by those skilled in the art, such a diagnosticassessment is usually not intended to be correct for all (i.e. 100%) ofthe patients to be identified. The term, however, as used herein refersto a diagnostic assessment that is correct such that a statisticallysignificant portion of patients can be identified (e.g. a cohort in acohort study). Statistically significance, as used herein, can bedetermined as known in the art using various well known statisticevaluation tools, for example determination of confidence intervals,p-value determination, Student's t-test, Mann-Whitney test etc. Detailsare found in Dowdy and Wearden, Statistics for Research, John Wiley &Sons, New York 1983. Exemplary confidence within the instant disclosureinclude intervals that are at least 90%, at least 95%, at least 97%, atleast 98% and/or at least 99%. The p-values are, for example, 0.1, 0.05,0.01, 0.005, and/or 0.0001. In exemplary embodiments of the instantdisclosure, at least 60%, at least 70%, at least 80% or at least 90% ofthe patients of a population can be properly identified by the method ofthe present disclosure.

In general, for determining the respective levels allowing to establishthe desired diagnosis in accordance with the respective embodiment ofthe present disclosure, (“threshold”, “reference level”), theamount(s)/level(s) or amount ratios of the respective peptide orpeptides are determined in appropriate patient groups. As stated before,the reference value is may be determined by multiplying an earlierdetermined IL-6 level (e.g. a baseline IL-6 level) in a given patient,for example in the subject patient, by a factor defined elsewhere herein(e.g. a factor of at least about 50 or at least about 100, or at leastabout 500 or at least about 1000). In some exemplary embodiments, basedon retrospective clinical studies, like the one described in theExamples, where the outcome (no SIRS vs. SIRS vs. sepsis) is analyzedand compared to the changes of the level of IL-6 in patients over time,it is easily possible to statistically verify which “factors” are to bemultiplied with a subject patient's baseline IL-6 level in order toestablish a reference value which is associated with a specific riskprobability of developing or suffering from SIRS or sepsis.

The reference level may also be calculated by multiplying the IL-6baseline level, by a factor of at least about 50, and in someembodiments by a factor of at least about 100, a factor of at leastabout 500, or even a factor of at least about 1000. For example, if theasymptomatic patient presenting to the physician or the hospital with abaseline IL-6 level of 10 pg/ml, the calculated reference level may be(according to some embodiments) 500 pg/ml (e.g., a 50-fold increase), 1ng/ml (e.g., a 100-fold increase), 5 ng/ml (a 500-fold increase), oreven 10 ng (a 1000-fold increase). In such a patient, a level of IL-6detected after subjecting the patient to a treatment (for example, asevere surgery or other invasive procedure) above the indicatedreference levels indicates that the patient is at high risk to developor suffer from SIRS.

As used herein, the “baseline level” encompasses at least one IL-6 levelobtained (i) when the patient presents to the physician, the emergencyunit, the hospital, the intensive care unit, the surgeon or theanaesthesiologist, (ii) before the therapy is initiated, e.g. beforesurgery, (iii) during the therapy, e.g. during surgery, and/or (iv)after the treatment has been completed, e.g. after the surgery has beencompleted. Also encompassed by the term is a baseline level which isdetermined after subjecting the patient to (e.g. an invasive) atreatment, or example within about 24 hours, (in some cases within about15, about 12 hours, or about 6 hours after treatment). Alternatively,the baseline level is calculated by determining the median or averageIL-6 level among the samples taken up to, but not including, a samplewhere the IL-6 level increased by more than about 20-fold, by more thanabout 30-fold, by more than about 50-fold, by more than about 50-fold,by more than about 100-fold, or by more than about 500-fold, relative tosaid median or average IL-6 level.

The diagnosis and detection of the risk of suffering from or developingSIRS or sepsis can be carried out by determining the respectiveparameters, preferably by measuring the level of IL-6, using validatedanalytical methods. The results which are obtained are collected andanalyzed by statistical methods known to the person skilled in the art.

Optionally, the reference values are established in accordance with thedesired probability of suffering from or being at risk to suffer ordevelop the disease. For example, it may be useful to choose the medianfactor to be multiplied with the baseline level from the 60th, 70th,80th, 90th, 95th or even the 99th percentile of the healthy and/ornon-healthy patient collective, in order to establish the referencelevel(s).

A reference level serving as a threshold may be derived from the upperlimit of normal (ULN), i.e. the upper limit of the physiological amountto be found in a population. The ULN for a given population of subjectscan be determined by various well known techniques. A suitable techniquemay be to determine the median of the population for the peptide orpolypeptide amounts to be determined in the method of the presentdisclosure.

A reference level of a diagnostic marker can be established andconfirmed, and the level of the marker in a patient sample can simply becompared to the reference level. The sensitivity and specificity of adiagnostic and/or prognostic test depends on more than just theanalytical “quality” of the test-they also depend on the definition ofwhat constitutes an abnormal result. In practice, Receiver OperatingCharacteristic curves, or “ROC” curves, are typically calculated byplotting the value of a variable versus its relative frequency in“normal” and “disease” populations. For any particular parameter of thedisclosure, a distribution of marker levels for subjects with andwithout a disease will likely overlap. Under such conditions, a testdoes not absolutely distinguish normal from disease with 100% accuracy,and the area of overlap indicates where the test cannot distinguishnormal from disease or high risk from low risk to suffer from or developthe disease. A threshold (cut-off, reference level) is selected, abovewhich (or below which, depending on how a marker changes with thedisease) the test is considered to be abnormal and below which the testis considered to be normal. The area under the ROC curve is a measure ofthe probability that the perceived measurement will allow correctidentification or prediction of the risk of onset or development of acondition. ROC curves can be used even when test results do notnecessarily give an accurate number. As long as one can rank results,one can create an ROC curve. For example, results of a test on “disease”samples might be ranked according to degree of the disease (say l=low,2=normal, and 3=high) or probability of suffering from or developing thedisease (say l=low, 2=normal, and 3=high). This ranking can becorrelated to results in the “normal” population, and a ROC curvecreated. These methods are well known in the art., for example asdiscussed in Hanley et al, Radiology 1982;143: 29-36, the disclosure ofwhich is hereby incorporated by reference in its entirety.

In certain embodiments, markers and/or marker panels are selected toexhibit at least about 70% sensitivity, and in some cases at least about80% sensitivity, at least about 85% sensitivity, at least about 90%sensitivity, and even at least about 95% sensitivity, combined with atleast about 70% specificity, but in some cases as high as at least about80% specificity, at least about 85% specificity, at least about 90%specificity, and even at least about 95% specificity. In particularembodiments, both the sensitivity and specificity are at least about75%, or at least about 80%, or even at least about 85%, about 90%, andeven at least about 95%.

In other embodiments, a positive likelihood ratio, negative likelihoodratio, or odds ratio is used as a measure of a test's ability to predictrisk or diagnose a disease. In the case of a positive likelihood ratio,a value of 1 indicates that a positive result is equally likely amongsubjects in both the “diseased” and “control” groups; a value greaterthan 1 indicates that a positive result is more likely in the diseasedgroup; and a value less than 1 indicates that a positive result is morelikely in the control group. In the case of a negative likelihood ratio,a value of 1 indicates that a negative result is equally likely amongsubjects in both the “diseased” and “control” groups; a value greaterthan 1 indicates that a negative result is more likely in the testgroup; and a value less than 1 indicates that a negative result is morelikely in the control group. In certain embodiments, markers and/ormarker panels are selected to exhibit a positive or negative likelihoodratio of at least about 1.5 or more or about 0.67 or less, in some casesat least about 2 or more or about 0.5 or less, and in still others atleast about 5 or more or about 0.2 or less, and in even others at leastabout 10 or more or about 0.1 or less, and in others at least about 20or more or about 0.05 or less. The term “about” in this context refersto +/−5% of a given measurement.

In the case of an odds ratio, a value of 1 indicates that a positiveresult is equally likely among subjects in both the “diseased” and“control” groups; a value greater than 1 indicates that a positiveresult is more likely in the diseased group; and a value less than 1indicates that a positive result is more likely in the control group. Incertain embodiments, markers and/or marker panels may be selected toexhibit an odds ratio of at least about 2 or more or about 0.5 or less,at least about 3 or more or about 0.33 or less, at least about 4 or moreor about 0.25 or less, at least about 5 or more or about 0.2 or less,and even at least about 10 or more or about 0.1 or less. The term“about” in this context refers to +/−5% of a given measurement.

Panels may comprise at least one additional marker; both specificmarkers of a disease (e.g., markers that are increased or decreased inbacterial infection, but not in other disease states) and/ornon-specific markers (e.g., markers that are increased or decreased dueto inflammation, regardless of the cause; markers that are increased ordecreased due to changes in hemostasis, regardless of the cause, etc.).While certain markers may not be individually definitive in the methodsdescribed herein, a particular “fingerprint” pattern of changes may, ineffect, act as a specific indicator of disease state. As discussedabove, that pattern of changes may be obtained from a single sample, ormay optionally consider temporal changes in one or more members of thepanel (or temporal changes in a panel response value).

In some embodiments of the methods of the present disclosure,

-   -   i) a level of IL-6 or a variant thereof determined in step a) at        or above to the reference level is indicative of the patient to        be at a high risk to suffering or developing SIRS; and    -   ii) a level of IL-6 or a variant thereof determined in step a)        below the reference level is indicative of the patient to be at        a low risk to suffering or developing SIRS.

It has been observed that the IL-6 concentration differs significantlyamong asymptomatic patients. For example, the following factorscontribute to this inter-individual differences: recent alcoholconsumption, physical exercise, stress, recent history of infections,injury and in acute hyperglycaemia. Accordingly, in some embodiments ofthe method of the present disclosure, the IL-6 reference level isdetermined on a patient-by-patient basis.

Patients displaying IL-6 levels above the reference level may also beclosely monitored for the onset of clinical signs and symptoms of SIRSor sepsis. The patients may be subjected to monitoring of the followingparameters: IL-6 levels, white blood cell count (WBC) and determinationof immature leucocyte forms, body temperature, heart-rate, respiratoryrate, collection of microbiological specimens from drainages, serum,tracheobronchial secretions and urine and daily chest roentgenogram.Control endoscopies and radiological workup by ultrasound and CT-scanmay also be warranted.

The patients may also be treated as follows: initiation of broadspectrum antibiotic treatment, antifungal treatment. In case of doubt,redo surgery shall be scheduled liberally.

In another aspect of the present disclosure it is provided a method ofmonitoring an asymptomatic patient the risk to develop or suffer fromSIRS or sepsis, comprising the steps of:

-   -   a) determining the level of IL-6 or a variant thereof in a        sample from the patient;    -   b) comparing the level of IL-6 or a variant thereof determined        in step a) to a reference level; and    -   c) recommending, deciding on, initiating, continuing, modulating        or discontinuing the SIRS or sepsis therapy for the patient        based on comparison in step c).

Diagnostic and therapeutic consequences of the above monitoring include:Microbiological specimens from serum, drainages, tracheobronchialsecretions, and urine, endoscopic controls, radiological imagingprocedures as are ultrasound, chest roentgenograms and CT-scans,initiation of broad spectrum antibiotic treatment or change ofpre-existent antibiotic treatment to a more efficient one, antifungaltreatment. In case of doubt, redo surgery shall be scheduled liberally.

In case the level of IL-6 or a variant thereof is higher than thereference level, the sample or an additionally collected sample issubjected to an assay to identify the infectious organism, e.g. thebacteria, fungus, etc., contained in the sample. Such assays aregenerally known in the field and are relevant to the diagnosis ofsepsis.

In another aspect of the present disclosure it is provided a method ofpredicting the risk of mortality in an asymptomatic patient, comprisingthe steps of

-   -   a) determining the level of IL-6 or a variant thereof in a        sample from the patient;    -   b) comparing the level of IL-6 or a variant thereof determined        in step a) to a reference level;    -   c) predicting the risk of mortality for the patient based on        comparison in step b).

Unless specified differently, the definitions and embodiments describedwith respect to the method for detection or diagnosis of a risk tosuffer from or develop SIRS, above, also apply mutatis mutandis to thepresent aspect of the disclosure.

According to yet another aspect of the disclosure it is provided adevice adapted for detection or diagnosis of a risk to suffer from ordevelop SIRS, according to a method described above, comprising:

-   -   a) a first analyzing unit comprising a detection means for IL-6        or a variant thereof, wherein the analyzing unit is adapted for        determining the level of the IL-6 detected by the detection        means;    -   b) an evaluation unit comprising a computer comprising tangibly        embedded a computer program code for carrying out the comparison        of the determined amount obtained from the first analyzing unit        with a suitable data base comprising a corresponding reference        level as specified above; wherein the first analyzing unit and        the evaluation units are operatively linked to each other.

In some embodiments, the device further comprises means for outputtingthe required diagnosis and treatment and/or prevention on the basis ofthe diagnosis or risk prediction of the patient to suffer from ordevelop SIRS or sepsis. In some embodiments, the device furthercomprises means for outputting the progress and/or response to atreatment and/or therapy of SIRS or sepsis.

According to yet another aspect of the disclosure it is provided a kitadapted for carrying out the method describe above, comprising:

-   -   means for determining the level of IL-6 or a variant thereof;    -   means for comparing the determined level of IL-6 or a variant        thereof with reference level; and    -   instructions for carrying out the method.

The term “kit” as used herein refers to a collection of theaforementioned compounds, means or reagents of the present disclosurewhich may or may not be packaged together. The components of the kit maybe comprised by separate vials (i.e. as a kit of separate parts) orprovided in a single vial. Moreover, it is to be understood that the kitof the present disclosure is to be used for practising the methodsreferred to herein above. According to some embodiments, all componentsare provided in a ready-to-use manner for practising the methodsreferred to above. Further, the kit may contain instructions forcarrying out the said methods. The instructions can be provided by auser's manual in paper or electronic form for example. For example, themanual may comprise instructions for interpreting the results obtainedwhen carrying out the aforementioned methods using the kit of thepresent disclosure.

According to yet another aspect of the disclosure it is provided acomputer program comprising computer program code which is suitable forcarrying out a method of the disclosure when the computer program is runon a computer. In another aspect of the disclosure it is provided acomputer readable medium with a computer program of the disclosurestored thereon. In yet another aspect of the disclosure it is provided acomputer program product with a computer program of the disclosurestored thereon. For example, the computer program further comprisesmeans for outputting the required prevention and/or therapy on the basisof the diagnosed disease, the means being stored on a computer readablemedium.

Unless specified differently, the definitions and preferred embodimentsdescribed with respect to the method detection or diagnosis of asystemic inflammatory response syndrome (SIRS), or for detection ordiagnosis of a risk to suffer from or develop SIRS, above, also apply tothe present aspect of the disclosure.

In another aspect of the disclosure it is provided a kit adapted forcarrying out the method of the present disclosure, comprising:

-   -   i) means for determining the level of IL-6 or a variant thereof;    -   ii) means for comparing the determined level of IL-6 or a        variant thereof with reference levels; and optionally    -   iii) instructions for carrying out the method.

In another aspect of the disclosure, it is provided a method fordetection or diagnosis of a systemic inflammatory response syndrome(SIRS) or sepsis, or for detection or diagnosis of a risk to suffer fromor develop SIRS, in an asymptomatic patient, comprising the steps of:

-   -   a) determining the level of CRP in a sample from the patient;    -   b) comparing the level of CRP determined in step a) to a        reference level; and    -   c) detecting or diagnosing SIRS, or detecting or diagnosing a        risk to develop SIRS;    -   wherein the sample is isolated at least 2 times at short        intervals and steps a) and b) are repeated for each sample.

In another aspect of the present disclosure it is provided a method fordetection or diagnosis of the risk to develop or suffer from a systemicinflammatory response syndrome (SIRS) or sepsis, in an asymptomaticpatient, comprising the steps of:

-   -   a) determining the level of procalcitonin in a sample from the        patient;    -   b) comparing the level of procalcitonin determined in step a) to        a reference level; and    -   c) detecting or diagnosing the risk to develop or suffer from a        systemic inflammatory response syndrome (SIRS) or sepsis;    -   wherein a sample is isolated at least 2 times at short intervals        and steps a) and b) are repeated for each sample.

In another aspect of the disclosure, it is provided an antibody selectedfrom an anti-IL-2 antibody, an anti-IL-3 antibody, an anti-IL-4antibody, an anti-IL-5 antibody, and an anti-IL-6 antibody.

Illustrative Embodiments

The following comprises a list of illustrative embodiments according tothe instant disclosure which represent various embodiments of theinstant disclosure. These illustrative embodiments are not intended tobe exhaustive or limit the disclosure to the precise forms disclosed,but rather, these illustrative embodiments are provided to aide infurther describing the instant disclosure so that others skilled in theart may utilize their teachings.

-   -   1. A method for detection or diagnosis of the risk to develop or        suffer from a systemic inflammatory response syndrome (SIRS) or        sepsis, in an asymptomatic patient, comprising the steps of:        -   a) determining the level of IL-6 or a variant thereof in a            sample from the patient;        -   b) comparing the level of IL-6 or a variant thereof            determined in step a) to a reference level;        -   c) detecting or diagnosing the risk to develop or suffer            from a systemic inflammatory response syndrome (SIRS) or            sepsis,            wherein a sample is isolated at least 2 times at short            intervals ranging from about 15 minutes to about 12 hours            and steps a) and b) are repeated for each sample.    -   2. The method of 1, wherein the asymptomatic patient is a        patient who displays less than 2, preferably less than 1 symptom        of the following a) to d), preferably of the following a) to e):    -   a) white blood cell count of more than about 12,000/μ/L or less        than about 4000/μ/L,    -   b) a body temperature of more than about 38° C. or less than        about 36° C.,    -   c) a heart rate of more than about 90 beats/minute or a partial        pressure of CO₂ of less than about 32 mm Hg, and    -   d) a respiratory rate of more than about 20 breaths/minute,    -   e) more than about 10% immature white blood cells among the        counted white blood cells, optionally the patient does not        display a diagnosed infection.

3. The method of 1 or 2, wherein the short interval ranges from about 1to 6 hours, preferably from about 1 hour to about 3 hours.

4. The method according to any one of 1 to 3, wherein

-   -   i) a level of IL-6 or a variant thereof determined in step a)        above to the reference level is indicative of the patient to be        at high risk of suffering or developing SIRS or sepsis; and    -   ii) a level of IL-6 or a variant thereof determined in step a)        below the reference level is indicative of the patient to be at        low risk of suffering or developing SIRS or sepsis.

5. The method according to any one of 1 to 4, wherein the referencelevel is obtained by multiplying a baseline level of IL-6 or a variantthereof by a factor of at least about 50, preferably by a factor of atleast about 100, more preferably by a factor of at least about 500, mostpreferably by a factor of at least about 1000.

6. The method of any one of 1 to 5, wherein IL-6 is an IL-6 which can bebound by murine anti-IL-6 monoclonal antibody M-BE8 or M-23C7.

7. The method of any one of 1 to 6, wherein the a level of IL-6 or avariant thereof in the sample is determined by

-   -   i) an antibody which binds to IL-6, or by a fragment or variant        thereof;    -   ii) an antibody which specifically binds to IL-6, or a fragment        or variant thereof;    -   iii) an antibody which binds to IL-6 which can be bound by        murine anti-IL-6 monoclonal antibody M-BE8 or M-23C7, or by a        fragment, or variant thereof; or    -   iv) a murine anti-IL-6 monoclonal antibody M-BE8 or M-23C7, or        by a fragment or variant thereof.

8. The method of any one of 1 to 7, wherein CRP is determined instead ofIL-6 and the method is used to diagnose SIRS or sepsis.

9. The method of any one of 1 to 8, wherein the asymptomatic patient isa patient selected from the group of a trauma patient, a patient withburns, a patient undergoing an treatment, a patient undergoing aninvasive treatment, a patient undergoing a surgical intervention.

10. The method of 9, wherein the patient is undergoing an invasivetreatment and the sample is taken at least once before the treatment andat least once after the treatment.

11. The method according to any one of 1 to 10, wherein the method

-   -   i) further comprises a step of collecting a sample from the        patient by a minimal-invasive step,    -   ii) excludes a surgical step of collecting a sample, or    -   iii) is an in vitro method.

12. A method of monitoring an asymptomatic patient for the onset of SIRSor for assessing the risk to develop or suffer from SIRS, comprising thesteps of

-   -   a) determining the level of IL-6 or a variant thereof in a        sample from the patient;    -   b) comparing the level of IL-6 or a variant thereof determined        in step a) to a reference level; and    -   c) recommending, deciding on, initiating, continuing, modulating        or discontinuing a SIRS therapy for the patient based on        comparison in step b).

13. The method of 12 wherein said reference level said reference levelis calculated by multiplying the IL-6 baseline level by a factor of atleast 50, said baseline level being obtained (i) when the patientpresents to the physician, the emergency unit, the hospital, theintensive care unit, the surgeon or the anaesthesiologist (ii) beforeand the therapy is initiated, (iii) during the therapy, and/or (iv)after the treatment has been completed.

14. The method of any of 1-13wherein the IL-6 or variant thereof has asequence identity to the human IL-6 molecule of at least 80% over theentire length of the human IL-6.

15. A kit adapted for carrying out the method of any of 1 to 14,comprising:

-   -   i) a means for determining the level of IL-6 or a variant        thereof;    -   ii) a means for comparing the determined level of IL-6 or a        variant thereof with reference levels; and optionally    -   iii) an instruction for carrying out the method.

16. Use of a means for detecting IL-6 or a variant thereof in a sampleof an asymptomatic patient, for early detecting or diagnosing the riskto develop or suffer from SIRS or sepsis, or for early detecting ordiagnosing SIRS or sepsis.

17. Use of an increase of the IL-6 level or the level of a variantthereof having a sequence identity to the human IL-6 molecule of atleast 80% over the entire length of the human IL-6 over time in samplesfrom an asymptomatic patient isolated at least 2 times at shortintervals ranging from 15 minutes +/−20% to 12 hours +/−20% for earlydetecting or diagnosing the risk to develop or suffer from SIRS orsepsis, or for early detecting or diagnosing SIRS or sepsis.

The following examples, sequence listing, and figures are provided forthe purpose of demonstrating various embodiments of the instantdisclosure and aiding in an understanding of the present disclosure, thetrue scope of which is set forth in the appended claims. These examplesare not intended to, and should not be understood as, limiting the scopeor spirit of the instant disclosure in any way. It should also beunderstood that modifications can be made in the procedures set forthwithout departing from the spirit of the disclosure.

EXAMPLES Patient Characteristics

48 patients, (36 males and 12 females), were included into the study.The mean age of the patients was 61 years, ranging from 19 to 87. Allpatients were admitted to a thoracic surgical division of theMedizinische Universitat Graz, Austria, for elective surgery. 37patients underwent lung surgery due to various carcinoma types, 9patients had oesophagoectomy because of an oesophagus carcinoma, 1patient underwent gastrectomy, and 1 patient underwent gastrectomy plusoesophagoectomy due to chemical burn. In all patients an antibiotictreatment was initiated in the morning of the day of surgery. Allpatients had two baseline level measurements before surgery todetermine, both, IL-6 and routine laboratory parameters.

Systemic Inflammatory Response Syndrome (SIRS) was diagnosed based onthe ACCP/SCCM Consensus Conference Definitions (1992/2003), i.e. a SIRSdiagnosed patients displayed at least 2 symptoms of the following:

-   -   a) white blood cell count of more than about 12,000/μ/L or less        than about 4000/μ/L;    -   b) a body temperature of more than about 38° C. or less than        about 36° C.;    -   c) a heart rate of more than about 90 beats/minute or a partial        pressure of CO₂ of less than about 32 mm Hg;    -   d) a respiratory rate of more than about 20 breaths/minute; and    -   e) more than about 10% immature white blood cells among the        counted white blood cells.

Out of the 48 patients 10 (21%) developed SIRS within the time window ofmonitoring of the study. Out of the 10 patients showing clinical signsof SIRS, three had in addition a positive blood culture which confirmedsepsis. Two of the cases responded to a change of antibiotic treatmentthough only in one patient the infection was confirmed by a positiveblood culture. 5 out of the 10 patients showed purely SIRS signs withoutinfection during the study period. One of the patients developed SIRSshortly after the end of the window of monitoring. In light of theresults discussed below this shows that IL-6 based diagnosis andprediction is not limited to sepsis but is also applicable to SIRS.

Material and Methods

IL-6 was determined using a sandwich ELISA immunoassay based on theRoche Cobas Elecsys IL-6 assay (Roche, Mannheim, Germany). In brief, 30μL of the plasma or serum sample were incubated with a biotinylatedmonoclonal IL-6-specific antibody. After addition of a monoclonalIL-6-specific antibody labeled with a ruthenium complex andstreptavidin-coated microparticles, the antibodies formed a sandwichcomplex with the antigen of the sample. The reaction mixture was thenaspirated into the measuring cell of the Elecsyselectrochemiluminescence device where the microparticles weremagnetically captured onto the surface of the electrode. Unboundsubstances were then removed with ProCell.

Application of a voltage to the electrode then induced chemiluminescentemission which was measured by a photomultiplier. Results weredetermined via a calibration curve which was instrument-specificallygenerated by 2-point calibration and a master curve provided via thereagent barcode. The determined IL-6 concentration was statisticallyanalyzed using standard statistical tests.

According to the study protocol the enrolled patients were monitoredregarding clinical and laboratory signs of SIRS starting with 2 baselinelevels taken at two time points before and during surgery. Aftersurgery, the patients were surveyed closely by taking blood samplestwice before, and every 6 hours during, 6 days after surgery. At everytime point when blood was taken the clinical status was recordedaccording to the established SIRS and sepsis criteria.

Results

Surprisingly, the IL-6 concentration values of the SIRS/sepsis patientsdiffered significantly from the median values of the patients who didnot develop SIRS. Even though the IL-6 levels increased in all patientsafter surgery, only the patients of the SIRS group displayed a largeincrease in IL-6 concentration relative to their respective baselinelevels, whereas the non-SIRS patients showed only an increase in IL-6concentration by up to 10-fold when compared to the respective baselinelevels (See, in general, the FIGS. 1-5).

Characteristics of some of the SIRS patients (see FIGS. 1 and 2) were asfollows:

Patient No. 1: male, age 62, oesophageal carcinoma, oesophagectomy.Routine laboratory parameters and IL-6 values before surgery wereinconspicuous (IL-6 baseline levels 14.3 and 2.9 pg/ml). On the firstday after surgery a baseline level of 6.4 pg/ml IL-6 was measured atnoon. The IL-6 level 6 hours later was 2.279.00 pg/ml. Though the valueswere decreasing over time, they stayed high until day 6 with valuesaround 500 pg/ml. Clinical SIRS/sepsis signs were only recognized firsttime on day 4, when a positive blood culture indicating a bacterialinfection and an increased body temperature >38° C. was documented. Onday 6 the heart rate was >90 beats/minute. This case demonstrates closeand frequent IL-6 monitoring allows for early diagnosis and detection ofa high risk to develop, or suffer from SIRS, well ahead of the onset ofclinical signs and symptoms supporting a diagnosis of SIRS or sepsis.

Patient No. 2: male, age 81, carcinoma at the esophagogastric junction.Before surgery the patient had a low leucocyte count. The secondbaseline level was actually <4000 white blood cells. On day 1 aftersurgery the patient had a body temperature of <36° C. and a breathingrate of >20 /minute. The two signs were taken as suggestive of SIRS. Atthe same time the IL-6 values which were at baseline 1, 1.6 pg/ml, andat baseline 2, 37.9 pg/ml, increased to 1400.00 pg/ml. Apart from theblood cell count, which was increased to >12 from day 2 until day 6,only the IL-6 values remained high (up to 500 pg/ml) until day 4 andthen slowly decreased. No positive blood culture result was obtained,i.e. no sepsis relevant infection was detected. This case demonstratesthat a level of IL-6 above the reference level allows for earlydiagnosis and detection of a high risk to develop or suffer from SIRS.

Patient No. 3: male, age 70, lung cancer, pneumonectomy. The patient hadinitially (baseline 1) an increased white blood cell count of >12,000but a normalized value at baseline 2 time point one week later. Thebaseline IL-6 levels were also within the normal range of 23.5 and 16.5pg/ml. During all time points after surgery no clinical signs other thanan elevated IL-6 level appeared which pointed to SIRS or sepsis. Afterday 2 the white blood cell count increased to levels of about 13,000 anddid not decrease. IL-6 increased after surgery to values of 200 to 300pg/ml with interim peaks of 600 pg/ml and even 850 pg/ml at onemeasuring point. On day 5 in the evening the IL-6 value suddenlyincreased to 3994.00 pg/ml, following a value of 148.6 pg/ml obtained 6hours earlier. Two days later, beyond the study protocol time line, thepatient developed SIRS criteria and developed a critical condition butcould be stabilized. No other laboratory or clinical parameter besideIL-6 had reflected the acute worsening of the patient. Accordingly, thetight monitoring of IL-6 surprisingly allowed for an early detection ofa high risk to develop SIRS or sepsis which allowed for an earlytherapeutic intervention.

Patient No. 4: female, age 50, chondrosarcoma, metastases in the lung,pneumonectomy. The WBC count at baseline, and during the entire studyperiod of 6 days, was in the normal range with a maximum value of 11,000at day 4. IL-6 increased during surgery (baseline 2) to a value of 282.7pg/ml. At the first measurement after surgery (day 1/1), IL-6 increasedto 1079 pg/ml and 6 hours later peaked at 2771 pg/ml. From that timepoint on, the values decreased continuously to values of about 70 pg/mlat the end of the study period. SIRS criteria (body temperature >38° C.and HR >90/min) appeared in this patient at the same time points as IL-6had its highest values which confirms that IL-6 is a reliable earlymarker of SIRS.

Patient No. 5: male,19 years, chemical burn of oesophagus and stomach.At baseline before surgery all parameter were in a normal range. IL-6increased slightly up to 67.4 pg/ml during surgery when the secondbaseline blood sample was taken. The next blood sample taken 6 hourslater (dayl/1) already showed an IL-6 value of more than 700 pg/ml whichfurther increased to more than 1000 pg/ml during the next days (day1/1-3/1). Clinical signs of SIRS (heart rate >100/min and breathrate >20/min) were registered from day 5/2 until the end of the studyperiod (day 6/2).

Patient No. 6: female, 60 years old, lobectomy after lung cancer. Atboth baseline levels all parameter were in the normal range. The firstIL-6 value after surgery (day 1/1) showed a rise to almost 500 pg/ml.IL-6 levels increased over the full study period showing levels between550 and 150 pg/ml. Leucocytes increased significantly and reached levelsof >20×10 from day 1/4 on, in conjunction with an increase in bodytemperature (>38° C.), whereby SIRS was registered. After two timepoints body temperature returned to normal again but increased once moreat day 6/2.

Patient No. 7: male, age 48 years, lung cancer, pneumectomy. Initially,at baseline all parameters were within a normal range. At the secondbaseline time point, the patient showed an increased heart rateof >100/min which persisted in nearly all study time points. IL-6increased significantly at day 1/1 to a value of almost 400 pg/ml andfell afterwards to moderately increased values of around 70 pg/ml overthe next 7 sampling time points. At day 4/1 IL-6 started to increaseagain reaching values of more than 500 pg/ml. Clinical signs of SIRS(heart rate >100/min and breath rate >20/min) were registered from day5/2 until the study end at day 6/2.

Characteristics of some of the non-SIRS patients (See FIGS. 1 and 3-5)were as follows:

38 out of the 48 patients who did not develop SIRS or sepsis, accordingto the definitions, displayed at least a single symptom of SIRS duringthe observation period but never displayed two or more, and accordinglythese patients were diagnosed as not suffering from SIRS. The baselineIL-6 values were (of non-SIRS patients) were comparable to the baselinevalues of the SIRS patients. After surgery all non-SIRS patients alsohad a slight increase in the IL-6, values some up to about 100 pg/ml,some up to 400, one with a single peak of 900 pg/ml, but they did notshow the drastic elevation of IL-6 levels observed in the SIRS patientgroup. The observed small increase in IL-6 levels in the non-SIRSpatients is within the range that one would expect, considering thesevereness of the conducted surgery. Based on the outcome (no onset ofSIRS or sepsis) in these patients, the data demonstrate that thepatients were at a low risk to suffer or develop SIRS or sepsis.

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

While this disclosure has been described as having an exemplary design,the present disclosure may be further modified within the spirit andscope of this disclosure. This application is therefore intended tocover any variations, uses, or adaptations of the disclosure using itsgeneral principles. Further, this application is intended to cover suchdepartures from the present disclosure as come within the known orcustomary practice in the art to which this disclosure pertains.

1. A method of providing a diagnosis of at risk for systemicinflammatory response syndrome (SIRS) or sepsis in an asymptomaticpatient, the method comprising the steps of: determining a level of IL-6or a variant thereof in a first sample of the asymptomatic patient, theIL-6 of variant thereof having a sequence identity to the human IL-6molecule of at least 80% over the entire length of the human IL-6;determining a level of the IL-6 or the variant thereof in a secondsample of the asymptomatic patient, the second sample being isolatedfrom the patient at a time interval of between 15 minutes +/−20% and 12hours +/−20% after the first sample; comparing the level of IL-6 or thevariant thereof determined in each of said steps of determining to areference level; and providing a diagnosis of at risk for systemicinflammatory response syndrome (SIRS) or sepsis if the IL-6 levelincreases over time.
 2. The method of claim 1, wherein the asymptomaticpatient is a patient who displays less than 2 symptoms of the symptomsselected from group consisting of: white blood cell count of more than12,000/μ/L +/−20% or less than about 4000/μ/L +/−20%; a body temperatureof more than 38° C. +/−20% or less than 36° C. +/−20%; a heart rate ofmore than 90 beats/minute +/−20% or a partial pressure of CO₂ of lessthan 32 mm Hg +/−20%; a respiratory rate of more than 20 breaths/minute+/−20%; and more than 10% immature white blood cells among the countedwhite blood cells.
 3. The method of claim 1, wherein the time intervalis of between 1 hour +/−20% and 3 hours +/−20%.
 4. The method of claim1, wherein said step of providing comprises providing a diagnosis of atrisk for systemic inflammatory response syndrome (SIRS) or sepsis if thelevel of IL-6 or the variant thereof determined in said steps ofdetermining is above to the reference level and a diagnosis of not atrisk for systemic inflammatory response syndrome (SIRS) or sepsis if thelevel of IL-6 or the variant thereof determined in said steps ofdetermining is below the reference level.
 5. The method of claim 1,wherein the reference level is obtained by multiplying a baseline levelof IL-6 or said variant thereof by a factor selected from the groupconsisting of: at least 50+/−20%; at least 100+/−20%; at least500+/−20%; and at least 1000+/−20%.
 6. The method of claim 1, whereinsaid steps of determining consist of contacting, in vitro, the first andsecond samples, respectively, with one of a murine anti-IL-6 monoclonalantibody M-BE8 and a M-23C7.
 7. The method of claim 1, wherein saidsteps of determining consist of contacting, in vitro, the first andsecond samples, respectively, with one of: an antibody which binds toIL-6 or a fragment or variant thereof; an antibody which binds to IL-6and can be bound by one of murine anti-IL-6 monoclonal antibody M-BE8,M-23C7, a fragment thereof, and a variant thereof; and a murineanti-IL-6 monoclonal antibody M-BE8 or M-23C7 or a fragment or variantthereof.
 8. The method of claim 1, wherein the asymptomatic patient is apatient selected from the group of a trauma patient, a patient withburns, a patient undergoing a treatment, a patient undergoing aninvasive treatment, and a patient undergoing a surgical intervention. 9.The method of claim 8, wherein the patient is undergoing an invasivetreatment, the first sample being isolated from the patient at leastonce before the treatment and the second sample being isolated from thepatient at least once after the treatment.
 10. The method of claim 1,wherein the method further comprises at least one of collecting thefirst sample from the patient by a minimal invasive step, excludes asurgical step of collecting the second sample, and is an in vitromethod.
 11. The method of claim 1, wherein said step of providingcomprises providing a diagnosis of at risk for systemic inflammatoryresponse syndrome (SIRS) or sepsis if the IL-6 level of the secondsample increases by greater than 10 fold over the IL-6 level of thefirst sample.
 12. A kit adapted for carrying out the method of claim 1,comprising: i) a means for determining the level of IL-6 or said variantthereof; ii) a means for comparing the determined level of IL-6 or saidvariant thereof, with reference levels; and iii) an instruction forcarrying out the method.
 13. A method of monitoring an asymptomaticpatient for onset of SIRS, comprising the steps of: determining a levelof IL-6 or a variant thereof in a sample of the asymptomatic patient,the IL-6 of variant thereof having a sequence identity to the human IL-6molecule of at least 80% over the entire length of the human IL-6;comparing the level of IL-6 or said variant thereof determined in saidstep of determining to a reference level; and based on said step ofcomparing, identifying said onset of SIRS if the level of IL-6determined in said step of determining is greater than the referencelevel.
 14. The method of claim 13, wherein the reference level isobtained by multiplying a baseline level of IL-6 or said variant thereofby a factor selected from the group consisting of: at least 100+/−20%;at least 500+/−20%; and at least 1000+/−20%.
 15. The method of claim 13,wherein said steps of determining consist of contacting, in vitro, thefirst and second samples, respectively, with one of a murine anti-IL-6monoclonal antibody M-BE8 and a M-23C7.